Straw – WGCNA

0.1 Импорт

Импорт phyloseq объекта и библиотек
Датасет - хроносерия разложения соломы - от фактор Day 10ти уровней

library(phyloseq)
library(tidyverse)
library(ggpubr)
library(ampvis2)
library(ANCOMBC)
library(heatmaply)
library(compositions)
library(igraph)
library(WGCNA)
require(DESeq2)
require(phyloseq)

ps.f <- readRDS("psf2")

0.2 functions

#phyloseq object to ampvis2 object
#https://gist.github.com/KasperSkytte/8d0ca4206a66be7ff6d76fc4ab8e66c6

phyloseq_to_ampvis2 <- function(physeq) {
  #check object for class
  if(!any(class(physeq) %in% "phyloseq"))
    stop("physeq object must be of class \"phyloseq\"", call. = FALSE)
  
  #ampvis2 requires taxonomy and abundance table, phyloseq checks for the latter
  if(is.null(physeq@tax_table))
    stop("No taxonomy found in the phyloseq object and is required for ampvis2", call. = FALSE)
  
  #OTUs must be in rows, not columns
  if(phyloseq::taxa_are_rows(physeq))
    abund <- as.data.frame(phyloseq::otu_table(physeq)@.Data)
  else
    abund <- as.data.frame(t(phyloseq::otu_table(physeq)@.Data))
  
  #tax_table is assumed to have OTUs in rows too
  tax <- phyloseq::tax_table(physeq)@.Data
  
  #merge by rownames (OTUs)
  otutable <- merge(
    abund,
    tax,
    by = 0,
    all.x = TRUE,
    all.y = FALSE,
    sort = FALSE
  )
  colnames(otutable)[1] <- "OTU"
  
  #extract sample_data (metadata)
  if(!is.null(physeq@sam_data)) {
    metadata <- data.frame(
      phyloseq::sample_data(physeq),
      row.names = phyloseq::sample_names(physeq), 
      stringsAsFactors = FALSE, 
      check.names = FALSE
    )
    
    #check if any columns match exactly with rownames
    #if none matched assume row names are sample identifiers
    samplesCol <- unlist(lapply(metadata, function(x) {
      identical(x, rownames(metadata))}))
    
    if(any(samplesCol)) {
      #error if a column matched and it's not the first
      if(!samplesCol[[1]])
        stop("Sample ID's must be in the first column in the sample metadata, please reorder", call. = FALSE)
    } else {
      #assume rownames are sample identifiers, merge at the end with name "SampleID"
      if(any(colnames(metadata) %in% "SampleID"))
        stop("A column in the sample metadata is already named \"SampleID\" but does not seem to contain sample ID's", call. = FALSE)
      metadata$SampleID <- rownames(metadata)
      
      #reorder columns so SampleID is the first
      metadata <- metadata[, c(which(colnames(metadata) %in% "SampleID"), 1:(ncol(metadata)-1L)), drop = FALSE]
    }
  } else
    metadata <- NULL
  
  #extract phylogenetic tree, assumed to be of class "phylo"
  if(!is.null(physeq@phy_tree)) {
    tree <- phyloseq::phy_tree(physeq)
  } else
    tree <- NULL
  
  #extract OTU DNA sequences, assumed to be of class "XStringSet"
  if(!is.null(physeq@refseq)) {
    #convert XStringSet to DNAbin using a temporary file (easiest)
    fastaTempFile <- tempfile(pattern = "ampvis2_", fileext = ".fa")
    Biostrings::writeXStringSet(physeq@refseq, filepath = fastaTempFile)
  } else
    fastaTempFile <- NULL
  
  #load as normally with amp_load
  ampvis2::amp_load(
    otutable = otutable,
    metadata = metadata,
    tree = tree,
    fasta = fastaTempFile
  )
}

#variance stabilisation from DESeq2

ps_vst <- function(ps, factor){
  

  
  diagdds = phyloseq_to_deseq2(ps, as.formula(paste( "~", factor)))              
  diagdds = estimateSizeFactors(diagdds, type="poscounts")
  diagdds = estimateDispersions(diagdds, fitType = "local") 
  pst <- varianceStabilizingTransformation(diagdds)
  pst.dimmed <- t(as.matrix(assay(pst))) 
  # pst.dimmed[pst.dimmed < 0.0] <- 0.0
  ps.varstab <- ps
  otu_table(ps.varstab) <- otu_table(pst.dimmed, taxa_are_rows = FALSE) 
  return(ps.varstab)
}

#WGCNA visualisation
#result - list class object with attributes:
# ps - phyloseq object
# amp - ampwis2 object
# heat - heatmap with absalute read numbers
# heat_rel - hetmap with relative abundances
# tree - phylogenetic tree with taxonomy

color_filt <- function(ps, df){
  
      library(tidyverse)
      library(reshape2)
      library(gridExtra)

  
  l = list()
  for (i in levels(df$module)){
    message(i)
    color_name <-  df %>% 
      filter(module == i) %>% 
      pull(asv) %>% 
      unique()
    ps.col <- prune_taxa(color_name, ps)
    amp.col <- phyloseq_to_ampvis2(ps.col)
    heat <- amp_heatmap(amp.col, tax_show = 60, 
              group_by = "Day", 
              tax_aggregate = "OTU",
              tax_add = "Genus", 
              normalise=FALSE, 
              showRemainingTaxa = TRUE)
    
    ps.rel  <-  phyloseq::transform_sample_counts(ps.col, function(x) x / sum(x) * 100)
    amp.r <- phyloseq_to_ampvis2(ps.rel)
    heat.rel <- amp_heatmap(amp.r, tax_show = 60, 
          group_by = "Day", 
          tax_aggregate = "OTU",
          tax_add = "Genus", 
          normalise=FALSE, 
          showRemainingTaxa = TRUE)
    
    tree <- ps.col@phy_tree
    taxa <- as.data.frame(ps.col@tax_table@.Data)
    p1 <- ggtree(tree) + 
              geom_tiplab(size=2, align=TRUE, linesize=.5) +
              theme_tree2()
    
    taxa[taxa == "Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium"] <- "Allorhizobium"
    taxa[taxa == "Burkholderia-Caballeronia-Paraburkholderia"] <- "Burkholderia"
  
    tx <- taxa %>% 
      rownames_to_column("id") %>% 
      mutate(id = factor(id, levels = rev(get_taxa_name(p1)))) %>% 
      dplyr::select(-c(Kingdom, Species, Order)) %>% 
      melt(id.var = 'id')

    p2 <- ggplot(tx, aes(variable, id)) + 
      geom_tile(aes(fill = value), alpha = 0.4) +
      geom_text(aes(label = value), size = 3) +
      theme_bw() + 
      theme(legend.position = "none",
             axis.ticks.x=element_blank(),
             axis.text.y=element_blank(),
             axis.ticks.y=element_blank(),
             axis.title.x = element_blank(),
            axis.title.y = element_blank()) 

    p <- ggpubr::ggarrange(p1, p2, widths = c(0.9, 1))
    l[[i]] <- list("ps" = ps.col, 
                   "amp" = amp.col,
                   "heat" = heat,
                   "heat_rel" = heat.rel,
                   "tree" = p,
                   "taxa" = knitr::kable(taxa))
                    
  }
  return(l)
}

detachAllPackages <- function() {

  basic.packages <- c("package:stats","package:graphics","package:grDevices","package:utils","package:datasets","package:methods","package:base")

  package.list <- search()[ifelse(unlist(gregexpr("package:",search()))==1,TRUE,FALSE)]

  package.list <- setdiff(package.list,basic.packages)

  if (length(package.list)>0)  for (package in package.list) detach(package, character.only=TRUE, force = TRUE)

}

plot_alpha_w_toc <- function(ps, group, metric) {
  
  require(phyloseq)
  require(ggplot2)
  
  ps_a <- prune_taxa(taxa_sums(ps) > 0, ps)
  
  er <- estimate_richness(ps_a)
  df_er <- cbind(ps_a@sam_data, er)
  df_er <- df_er %>% select(c(group, metric))
  stat.test <- aov(as.formula(paste0(metric, "~", group)), data = df_er) %>%
    rstatix::tukey_hsd()
  y <-  seq(max(er[[metric]]), length=length(stat.test$p.adj), by=max(er[[metric]]/20))

  plot_richness(ps_a, x=group, measures=metric) + 
    geom_boxplot() +
    geom_point(size=1.2, alpha=0.3) +
    ggpubr::stat_pvalue_manual(
      stat.test, 
      label = "p.adj.signif", 
      y.position = y) +
    theme_light() + 
    scale_color_brewer(palette="Dark2") +
    theme(axis.text.x = element_text(angle = 90),
          axis.title.x=element_blank()) +
    labs(y=paste(metric, "index")) 
}


# standart NMDS plot tool frop phyloseq with some additional aestatics 
# have stress value on plot - may work as fuck
beta_custom_norm_NMDS_elli_w <- function(ps, seed = 7888, normtype="vst", Color="What", Group="Repeat"){
  require(phyloseq)
  require(ggplot2)
  require(ggpubr)
  library(ggforce)
  
  
  ordination.b <- ordinate(ps, "NMDS", "bray")
  mds <- as.data.frame(ordination.b$points)
  p  <-  plot_ordination(ps,
                         ordination.b,
                         type="sample",
                         color = Color,
                         title="NMDS - Bray-Curtis",
                         # title=NULL,
                         axes = c(2,3) ) + 
    theme_bw() + 
    theme(text = element_text(size = 10)) + 
    geom_point(size = 3) +
    annotate("text",
    x=min(mds$MDS1) + abs(min(mds$MDS1))/7,
    y=max(mds$MDS2),
    label=paste0("Stress -- ", round(ordination.b$stress, 3))) +
    geom_mark_ellipse(aes_string(group = Group, label = Group),
                      label.fontsize = 10,
                      label.buffer = unit(2, "mm"),
                      label.minwidth = unit(5, "mm"),
                      con.cap = unit(0.1, "mm"),
                      con.colour='gray') +
    theme(legend.position = "none") +
    ggplot2::scale_fill_viridis_c(option = "H")
  
  return(p)
}


# alpha with aov + tukie post-hock - useless, but it looks pretty good
plot_alpha_w_toc <- function(ps, group, metric) {
  
  require(phyloseq)
  require(ggplot2)
  
  ps_a <- prune_taxa(taxa_sums(ps) > 0, ps)
  
  er <- estimate_richness(ps_a)
  df_er <- cbind(ps_a@sam_data, er)
  df_er <- df_er %>% select(c(group, metric))
  stat.test <- aov(as.formula(paste0(metric, "~", group)), data = df_er) %>%
    rstatix::tukey_hsd()
  y <- seq(max(er[[metric]]), length=length(stat.test$p.adj.signif[stat.test$p.adj.signif != "ns"]), by=max(er[[metric]]/20))
  
  plot_richness(ps_a, x=group, measures=metric) + 
    geom_boxplot() +
    geom_point(size=1.2, alpha=0.3) +
    stat_pvalue_manual(
      stat.test, 
      label = "p.adj.signif", 
      y.position = y,
      hide.ns=TRUE) +
    theme_light() + 
    scale_color_brewer(palette="Dark2") +
    theme(axis.text.x = element_text(angle = 90),
          axis.title.x=element_blank()) +
    labs(y=paste(metric, "index")) 
}

0.3 Alpha

p1 <- plot_alpha_w_toc(ps = ps.f, group = "Day", metric = "Observed")

## Note: Using an external vector in selections is ambiguous.
## ℹ Use `all_of(group)` instead of `group` to silence this message.
## ℹ See <https://tidyselect.r-lib.org/reference/faq-external-vector.html>.
## This message is displayed once per session.
## Note: Using an external vector in selections is ambiguous.
## ℹ Use `all_of(metric)` instead of `metric` to silence this message.
## ℹ See <https://tidyselect.r-lib.org/reference/faq-external-vector.html>.
## This message is displayed once per session.

p2 <- plot_alpha_w_toc(ps = ps.f, group = "Day", metric = "Shannon")
p3 <- plot_alpha_w_toc(ps = ps.f, group = "Day", metric = "InvSimpson")
ggarrange(p1, p2, p3, nrow = 1)

Add Group parameter to metadata -
- early - D01, D03, D05
- middle - D07, D08, D10
- late - D13, D14, D15

sample.data <- ps.f@sam_data %>% 
  data.frame() %>% 
  mutate(Group = if_else(Day %in% c("D01", "D03", "D05"), "early", 
                         if_else(Day %in% c("D07", "D08","D10"), "middle", "late"))) %>% 
  mutate(Group = factor(Group, levels=c("early", "middle","late"))) %>% 
  phyloseq::sample_data()

sample_data(ps.f) <- sample.data

p.observed <- plot_alpha_w_toc(ps = ps.f, group = "Group", metric = c("Observed")) + 
  theme(axis.title.y = element_blank())

p.shannon <- plot_alpha_w_toc(ps = ps.f, group = "Group", metric = c("Shannon")) + 
  theme(axis.title.y = element_blank())

p.simpson <- plot_alpha_w_toc(ps = ps.f, group = "Group", metric = c("InvSimpson")) + 
  theme(axis.title.y = element_blank())

ggpubr::ggarrange(p.observed, p.shannon, p.simpson, ncol = 3)

0.4 beta statistic

0.4.1 permanova

permanova - group significantlly different - dispersion between is more, than inside groups

dist <- phyloseq::distance(ps.f, "bray")
metadata <- as(sample_data(ps.f@sam_data), "data.frame")
vegan::adonis2(dist ~ Group, data = metadata)

## Permutation test for adonis under reduced model
## Terms added sequentially (first to last)
## Permutation: free
## Number of permutations: 999
## 
## vegan::adonis2(formula = dist ~ Group, data = metadata)
##          Df SumOfSqs      R2      F Pr(>F)    
## Group     2   3.2742 0.33893 8.2033  0.001 ***
## Residual 32   6.3861 0.66107                  
## Total    34   9.6604 1.00000                  
## ---
## Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

0.4.2 dynamic of beta diversity

Дистанция брей кертиса по шагам между каждыми днями.   (Например - все D15 против D14)   Может на эту картинку подвесить еще почвенное дыхание?

ps.f.r <- rarefy_even_depth(ps.f, rngseed = 777)

avg.r <- ps.f.r@otu_table %>% 
  as.data.frame() %>%
  vegan::avgdist(10)

avg <- ps.f@otu_table %>% 
  as.data.frame() %>%
  vegan::avgdist(10)

# avg %>%
#   as.matrix() %>%
#   as_tibble(rownames= "sample") %>%
#   pivot_longer(-sample) %>%
#   filter(sample < name) %>%
#   mutate(repeat_a = str_replace(sample, ".*-", ""),
#          repeat_b = str_replace(name, ".*-", ""), 
#          day_a = as.numeric(str_replace(sapply(strsplit(sample, "-"), `[`, 3), "D", "")),
#           day_b = as.numeric(str_replace(sapply(strsplit(name, "-"), `[`, 3), "D", "")),
#          diff = abs(day_a - day_b),
#          early = day_a < 10) %>% 
#   filter(repeat_a == repeat_b & diff < 10) %>%
#   group_by(diff, repeat_a, early) %>%
#   summarize(median = median(value)) %>%
#   ungroup() %>%
#   ggplot(aes(x=diff, y=median, color=early, group=paste0(repeat_a, early))) +
#   geom_line(size=0.25) +
#   geom_smooth(aes(group=early), se=FALSE, size=4) +
#   labs(x="Distance between time points",
#        y="Median Bray-Curtis distance") +
#   scale_x_continuous(breaks=1:9) +
#   scale_color_manual(name=NULL,
#                      breaks=c(TRUE, FALSE),
#                      values=c("blue", "red"),
#                      labels=c("Early", "Late")) +
#   guides(color = guide_legend(override.aes = list(size=1))) +
#   theme_classic()

avg.r %>%
  as.matrix() %>%
  as_tibble(rownames= "sample") %>%
  pivot_longer(-sample) %>%
  filter(sample < name) %>%
  mutate(repeat_a = str_replace(sample, ".*-", ""),
         repeat_b = str_replace(name, ".*-", ""), 
         day_a = as.numeric(str_replace(sapply(strsplit(sample, "-"), `[`, 3), "D", "")),
          day_b = as.numeric(str_replace(sapply(strsplit(name, "-"), `[`, 3), "D", ""))) %>% 
  mutate(day_a = as.numeric(as.factor(day_a) %>% forcats::fct_recode("2" = "3", "3" = "5", "4" = "7", "5" = "8", "6" = "10", "7" = "13", "8" = "14", "9" = "14", "10" = "15")),
         day_b = as.numeric(as.factor(day_b) %>% forcats::fct_recode("2" = "3", "3" = "5", "4" = "7", "5" = "8", "6" = "10", "7" = "12", "8" = "13", "9" = "14", "10" = "15")),
         diff = abs(day_a - day_b)) %>% 
  filter(diff == 1) %>% 
  mutate(day_b = as.factor(day_b) %>% forcats::fct_recode("D03" = "2", "D05" = "3","D07" = "4",  "D08" = "5", "D10" = "6", "D13" = "7", "D14" = "8", "D15" = "10", "D12" = "7")) %>% 
  ggplot(aes(x=day_b, y=value)) +
    geom_boxplot() +
  theme_bw() +
  labs(x="Time points",
       y="Bray-Curtis distance")

0.4.3 basic NMDS plot

IN WORK!!

beta_custom_norm_NMDS_elli_w(ps.f, C="Group", G="Day")

## Square root transformation
## Wisconsin double standardization
## Run 0 stress 0.1048129 
## Run 1 stress 0.1372731 
## Run 2 stress 0.1318495 
## Run 3 stress 0.1048129 
## ... Procrustes: rmse 0.00004856956  max resid 0.0001949092 
## ... Similar to previous best
## Run 4 stress 0.1048129 
## ... New best solution
## ... Procrustes: rmse 0.00001842645  max resid 0.00006948497 
## ... Similar to previous best
## Run 5 stress 0.1426236 
## Run 6 stress 0.1048204 
## ... Procrustes: rmse 0.001770292  max resid 0.007527799 
## ... Similar to previous best
## Run 7 stress 0.1064532 
## Run 8 stress 0.1064733 
## Run 9 stress 0.1048204 
## ... Procrustes: rmse 0.001767644  max resid 0.007510715 
## ... Similar to previous best
## Run 10 stress 0.1064532 
## Run 11 stress 0.1318495 
## Run 12 stress 0.1064733 
## Run 13 stress 0.1064733 
## Run 14 stress 0.1460032 
## Run 15 stress 0.1048204 
## ... Procrustes: rmse 0.001769848  max resid 0.007525048 
## ... Similar to previous best
## Run 16 stress 0.1064532 
## Run 17 stress 0.1372728 
## Run 18 stress 0.1048129 
## ... Procrustes: rmse 0.000002711789  max resid 0.0000100797 
## ... Similar to previous best
## Run 19 stress 0.1460029 
## Run 20 stress 0.146003 
## *** Solution reached

0.5 Splitting the dataset

Разделим датасет на две группы

1-я - в более чем 10% образцов должно быть хотя бы 10 ридов - эта группа пойдет в анализ далее

ps.inall <- phyloseq::filter_taxa(ps.f, function(x) sum(x > 10) > (0.1*length(x)), TRUE)
amp.inall <- phyloseq_to_ampvis2(ps.inall)
amp_heatmap(amp.inall,
            tax_show = 40,
            group_by = "Day",
            tax_aggregate = "OTU",
            tax_add = "Genus",
            normalise=FALSE,
            showRemainingTaxa = TRUE)

Вторая - оставшиеся, но более 100 ридов по всем образцам(далее - вылетающие мажоры(ВМ))
Остальные филотипы выкидываем из анализа

ps.exl  <- phyloseq::filter_taxa(ps.f, function(x) sum(x > 10) < (0.1*length(x)), TRUE)
ps.exl  <- prune_taxa(taxa_sums(ps.exl) > 100, ps.exl)
amp.exl <- phyloseq_to_ampvis2(ps.exl)
amp_heatmap(amp.exl,
            tax_show = 40,
            group_by = "Day",
            tax_aggregate = "OTU",
            tax_add = "Genus",
            normalise=FALSE,
            showRemainingTaxa = TRUE)

То же, но c относительной представленностью.

ps.per  <-  phyloseq::transform_sample_counts(ps.f, function(x) x / sum(x) * 100) 
ps.exl.taxa <- taxa_names(ps.exl)
ps.per.exl <- prune_taxa(ps.exl.taxa, ps.per)
amp.exl.r <- phyloseq_to_ampvis2(ps.per.exl)

amp_heatmap(amp.exl.r, tax_show = 60, 
      group_by = "Day", 
      tax_aggregate = "OTU",
      tax_add = "Genus", 
      round =  2, 
      normalise=FALSE, 
      showRemainingTaxa = TRUE)

amp_heatmap(amp.exl.r, tax_show = 60, 
      group_by = "Day", 
      tax_aggregate = "OTU",
      tax_add = "Genus", 
      round =  2, 
      normalise=FALSE, )

ВМ объединенные по родам. Относительная представленность.

amp_heatmap(amp.exl.r,
            tax_show = 30, 
            group_by = "Day", 
            tax_aggregate = "Genus",
            tax_add = "Phylum",
            tax_class = "Proteobacteria",
            round =  2,
            normalise=FALSE, 
            showRemainingTaxa = TRUE)

Если посмотреть, если как распределяются ВМ по дням - похоже распределение ВМ связано не только с особенностью отдельных мешочков, но и с чисто техническими особенностями - вылетающие значения вылетают в основном не с биологическими повторностями, а с техническими(красная линия - выбрана визуально)

p_box <- phyloseq::sample_sums(ps.per.exl) %>% 
  as.data.frame(col.names = "values") %>% 
  setNames(., nm = "values") %>% 
  rownames_to_column("samples") %>% 
  mutate(Day = sapply(strsplit(samples, "-"), `[`, 3)) %>% 
  ggplot(aes(x=Day, y=values, color=Day, fill = Day)) +
  geom_boxplot(aes(color=Day, fill = Day)) +
  geom_point(color = "black", position = position_dodge(width=0.2)) +
  geom_hline(yintercept = 10, colour = "red") +
  theme_bw() +
  theme(legend.position = "none")
     
p_box <- p_box + viridis::scale_color_viridis(option = "H", discrete = TRUE, direction=1, begin=0.1, end = 0.9, alpha = 0.5)

p_box + viridis::scale_fill_viridis(option = "H", discrete = TRUE, direction=1, begin=0.1, end = 0.9, alpha = 0.3)

0.6 WGCNA

0.6.1 clr

после clr нормализации / выглядит отвратительно попробуем нормализацию vst из DESeq2

otu.inall <- as.data.frame(ps.inall@otu_table@.Data) 
ps.inall.clr <- ps.inall
otu_table(ps.inall.clr) <- phyloseq::otu_table(compositions::clr(otu.inall), taxa_are_rows = FALSE)
data <- ps.inall.clr@otu_table@.Data %>% 
  as.data.frame()
rownames(data) <- as.character(ps.inall.clr@sam_data$Description)
powers <-  c(c(1:10), seq(from = 12, to=30, by=1))
sft <-  pickSoftThreshold(data, powerVector = powers, verbose = 5, networkType = "signed")

## pickSoftThreshold: will use block size 338.
##  pickSoftThreshold: calculating connectivity for given powers...
##    ..working on genes 1 through 338 of 338

## Warning: executing %dopar% sequentially: no parallel backend registered

##    Power SFT.R.sq  slope truncated.R.sq  mean.k. median.k.  max.k.
## 1      1    0.185  23.60         0.8790 168.0000 168.00000 172.000
## 2      2    0.199 -15.50         0.8970  87.6000  87.20000  95.100
## 3      3    0.518 -13.10         0.8580  47.3000  46.80000  55.900
## 4      4    0.390  -6.46         0.8050  26.5000  26.00000  34.500
## 5      5    0.262  -3.46         0.7820  15.4000  14.90000  22.300
## 6      6    0.232  -2.30         0.7770   9.2300   8.78000  15.000
## 7      7    0.251  -1.87         0.6990   5.7300   5.36000  10.500
## 8      8    0.319  -1.75         0.7570   3.6800   3.41000   7.620
## 9      9    0.527  -2.07         0.7770   2.4400   2.20000   5.890
## 10    10    0.648  -2.28         0.8490   1.6600   1.47000   4.690
## 11    12    0.827  -2.41         0.8970   0.8420   0.69300   3.190
## 12    13    0.793  -2.38         0.7930   0.6220   0.49000   2.700
## 13    14    0.277  -3.97         0.0914   0.4690   0.35500   2.320
## 14    15    0.282  -3.83         0.1000   0.3610   0.26100   2.010
## 15    16    0.894  -2.17         0.9030   0.2820   0.19800   1.770
## 16    17    0.905  -2.10         0.9080   0.2240   0.15000   1.560
## 17    18    0.922  -2.04         0.9240   0.1810   0.11500   1.390
## 18    19    0.927  -2.01         0.9230   0.1480   0.08820   1.250
## 19    20    0.938  -1.93         0.9370   0.1220   0.06780   1.130
## 20    21    0.946  -1.86         0.9470   0.1020   0.05260   1.020
## 21    22    0.315  -3.12         0.2220   0.0856   0.04140   0.926
## 22    23    0.303  -3.96         0.1560   0.0728   0.03270   0.844
## 23    24    0.307  -2.92         0.1940   0.0623   0.02590   0.772
## 24    25    0.303  -2.84         0.1800   0.0538   0.02060   0.708
## 25    26    0.921  -1.63         0.8990   0.0467   0.01650   0.657
## 26    27    0.892  -1.61         0.8650   0.0408   0.01320   0.622
## 27    28    0.155  -1.93        -0.0202   0.0359   0.01040   0.591
## 28    29    0.199  -2.77        -0.0228   0.0317   0.00841   0.563
## 29    30    0.199  -2.69        -0.0203   0.0282   0.00684   0.537

plot(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2], xlab="Soft Threshold (power)",ylab="Scale Free Topology Model Fit,signed R^2",type="n", main = paste("Scale independence"))
text(sft$fitIndices[,1], -sign(sft$fitIndices[,3])*sft$fitIndices[,2], labels=powers,cex=0.9,col="red")
abline(h=0.9,col="salmon")

0.6.2 vst

после vst нормализации

ps.varstab <- ps_vst(ps.inall, "Day")

data2 <- ps.varstab@otu_table@.Data %>% 
  as.data.frame()

rownames(data2) <- as.character(ps.varstab@sam_data$Description)
powers <-  c(seq(from = 1, to=10, by=0.5), seq(from = 11, to=20, by=1))
sft2 <-  pickSoftThreshold(data2, powerVector = powers, verbose = 5, networkType = "signed hybrid")

## pickSoftThreshold: will use block size 338.
##  pickSoftThreshold: calculating connectivity for given powers...
##    ..working on genes 1 through 338 of 338
##    Power SFT.R.sq slope truncated.R.sq mean.k. median.k. max.k.
## 1    1.0    0.409 -1.05          0.704  50.100  45.60000  97.80
## 2    1.5    0.578 -1.07          0.895  30.900  27.00000  71.20
## 3    2.0    0.723 -1.15          0.942  20.300  17.90000  53.60
## 4    2.5    0.767 -1.24          0.871  14.000  12.00000  41.70
## 5    3.0    0.809 -1.31          0.909  10.000   7.86000  33.40
## 6    3.5    0.872 -1.31          0.958   7.380   5.39000  27.30
## 7    4.0    0.888 -1.30          0.968   5.580   3.76000  22.60
## 8    4.5    0.858 -1.36          0.910   4.320   2.67000  19.00
## 9    5.0    0.892 -1.36          0.943   3.400   1.94000  16.20
## 10   5.5    0.899 -1.38          0.954   2.720   1.41000  13.90
## 11   6.0    0.896 -1.39          0.962   2.210   1.07000  12.10
## 12   6.5    0.897 -1.33          0.962   1.820   0.84700  10.60
## 13   7.0    0.910 -1.30          0.970   1.520   0.65400   9.30
## 14   7.5    0.914 -1.31          0.966   1.280   0.51500   8.25
## 15   8.0    0.913 -1.28          0.954   1.090   0.41500   7.36
## 16   8.5    0.905 -1.28          0.937   0.938   0.34000   6.59
## 17   9.0    0.895 -1.30          0.913   0.813   0.27800   5.99
## 18   9.5    0.878 -1.33          0.900   0.709   0.23200   5.49
## 19  10.0    0.886 -1.34          0.894   0.624   0.19100   5.07
## 20  11.0    0.911 -1.31          0.928   0.491   0.12500   4.35
## 21  12.0    0.890 -1.29          0.894   0.396   0.08590   3.79
## 22  13.0    0.910 -1.26          0.918   0.325   0.06120   3.33
## 23  14.0    0.882 -1.25          0.856   0.272   0.04420   2.95
## 24  15.0    0.889 -1.19          0.864   0.230   0.03230   2.63
## 25  16.0    0.913 -1.18          0.893   0.198   0.02240   2.35
## 26  17.0    0.930 -1.16          0.912   0.172   0.01620   2.20
## 27  18.0    0.955 -1.19          0.947   0.151   0.01190   2.09
## 28  19.0    0.908 -1.21          0.896   0.134   0.00896   1.99
## 29  20.0    0.916 -1.21          0.906   0.120   0.00649   1.91

plot(sft2$fitIndices[,1], -sign(sft2$fitIndices[,3])*sft2$fitIndices[,2], xlab="Soft Threshold (power)",ylab="Scale Free Topology Model Fit,signed R^2",type="n", main = paste("Scale independence"))
text(sft2$fitIndices[,1], -sign(sft2$fitIndices[,3])*sft2$fitIndices[,2], labels=powers,cex=0.9,col="red")
abline(h=0.9,col="salmon")

# WGCNA, as old and not supported
detachAllPackages()
library(WGCNA)


net3 <- WGCNA::blockwiseModules(data2,
                          power=5.5,
                          TOMType="signed",
                          networkType="signed hybrid",
                          nThreads=0)


mergedColors2 <-  WGCNA::labels2colors(net3$colors, colorSeq = c("salmon", "darkgreen", "cyan", "red", "blue", "plum"))

plotDendroAndColors(
  net3$dendrograms[[1]],
  mergedColors2[net3$blockGenes[[1]]],
  "Module colors",
  dendroLabels = FALSE,
  hang = 0.03,
  addGuide = TRUE,
  guideHang = 0.05)

0.6.2.1 Импорт 2

library(phyloseq)
library(tidyverse)
library(ggpubr)
library(ampvis2)
library(heatmaply)
library(WGCNA)
library(phyloseq)
library(ggtree)
library(tidyverse)
library(KneeArrower)

modules_of_interest = mergedColors2 %>% 
  unique()

module_df <- data.frame(
  asv = names(net3$colors),
  colors = mergedColors2
)

# module_df[module_df == "yellow"] <- "salmon"

submod <-  module_df %>%
  subset(colors %in% modules_of_interest)

row.names(module_df) = module_df$asv

subexpr = as.data.frame(t(data2))[submod$asv,]

submod_df <- data.frame(subexpr) %>%
  mutate(
    asv = row.names(.)
  ) %>%
  pivot_longer(-asv) %>%
  mutate(
    module = module_df[asv,]$colors
  )

submod_df <- submod_df %>% 
  mutate(name = gsub("\\_.*","",submod_df$name)) %>% 
  group_by(name, asv) %>% 
  summarise(value = mean(value), asv = asv, module = module) %>% 
  relocate(c(asv, name, value, module)) %>% 
  ungroup() %>% 
  mutate(module = as.factor(module))

p <- submod_df %>% 
  ggplot(., aes(x=name, y=value, group=asv)) +
  geom_line(aes(color = module),
            alpha = 0.2) +
  theme_bw() +
  theme(
    axis.text.x = element_text(angle = 90),
    legend.position = "none") +
  facet_grid(rows = vars(module)) +
  labs(x = "treatment",
       y = "normalized expression")

p + scale_color_manual(values = levels(submod_df$module))

0.6.2.2 ordination

UNIFRAC - колбаска сужается

ps.inall.col <- ps.inall
df <- module_df %>% 
  rename("id" = "asv")
df <- df %>% 
  dplyr::select(-"id") %>% 
  mutate(colors = as.factor(colors))
taxa <- as.data.frame(ps.inall@tax_table@.Data)
tx <- cbind(taxa, df)
tx$colors <- factor(tx$colors, levels = c("salmon", "darkgreen", "cyan", "red", "blue", "plum"))
tax_table(ps.inall.col) <- tax_table(as.matrix(tx))
ord <- ordinate(ps.inall.col, "NMDS", "unifrac")

## Run 0 stress 0.0685455 
## Run 1 stress 0.05568715 
## ... New best solution
## ... Procrustes: rmse 0.04076806  max resid 0.2160826 
## Run 2 stress 0.06784429 
## Run 3 stress 0.05628488 
## Run 4 stress 0.05568719 
## ... Procrustes: rmse 0.0001769095  max resid 0.0006062451 
## ... Similar to previous best
## Run 5 stress 0.07937976 
## Run 6 stress 0.07544596 
## Run 7 stress 0.05628489 
## Run 8 stress 0.05568719 
## ... Procrustes: rmse 0.00002559041  max resid 0.00008856774 
## ... Similar to previous best
## Run 9 stress 0.05568712 
## ... New best solution
## ... Procrustes: rmse 0.00002106782  max resid 0.00006934972 
## ... Similar to previous best
## Run 10 stress 0.06740137 
## Run 11 stress 0.06740136 
## Run 12 stress 0.06784421 
## Run 13 stress 0.06784436 
## Run 14 stress 0.06854546 
## Run 15 stress 0.07953205 
## Run 16 stress 0.05568719 
## ... Procrustes: rmse 0.00004255519  max resid 0.0001401031 
## ... Similar to previous best
## Run 17 stress 0.06740138 
## Run 18 stress 0.06728216 
## Run 19 stress 0.05568718 
## ... Procrustes: rmse 0.0000373923  max resid 0.0001312747 
## ... Similar to previous best
## Run 20 stress 0.07888487 
## *** Solution reached

plot_ordination(ps.inall.col, ord, type = "species", color = "colors") + 
  scale_color_manual(values =  c("salmon", "darkgreen", "cyan", "red", "blue", "plum")) +
  theme_bw() +
  theme(legend.position = "none")

Bray - колбаска равномерна
Поздние стадии слева - при этом ранние кластеры накладываются, поздние разделены Что влияет на ось2? Явно есть какой-то паттерн.

ord <- ordinate(ps.inall.col, "NMDS", "bray")

## Square root transformation
## Wisconsin double standardization
## Run 0 stress 0.09036937 
## Run 1 stress 0.0903694 
## ... Procrustes: rmse 0.00001927814  max resid 0.00007086537 
## ... Similar to previous best
## Run 2 stress 0.09024752 
## ... New best solution
## ... Procrustes: rmse 0.003386048  max resid 0.01505777 
## Run 3 stress 0.09036938 
## ... Procrustes: rmse 0.003390695  max resid 0.01505826 
## Run 4 stress 0.09036937 
## ... Procrustes: rmse 0.003386035  max resid 0.01507802 
## Run 5 stress 0.09036937 
## ... Procrustes: rmse 0.003386432  max resid 0.01505862 
## Run 6 stress 0.1257473 
## Run 7 stress 0.09036937 
## ... Procrustes: rmse 0.003385691  max resid 0.01507976 
## Run 8 stress 0.1268919 
## Run 9 stress 0.09036944 
## ... Procrustes: rmse 0.003410952  max resid 0.01523599 
## Run 10 stress 0.09024752 
## ... New best solution
## ... Procrustes: rmse 0.00003844186  max resid 0.0001638862 
## ... Similar to previous best
## Run 11 stress 0.09036937 
## ... Procrustes: rmse 0.003380784  max resid 0.0150593 
## Run 12 stress 0.09036938 
## ... Procrustes: rmse 0.003379871  max resid 0.01508219 
## Run 13 stress 0.09036939 
## ... Procrustes: rmse 0.003385498  max resid 0.01512324 
## Run 14 stress 0.1358024 
## Run 15 stress 0.09024754 
## ... Procrustes: rmse 0.00003794727  max resid 0.0001665169 
## ... Similar to previous best
## Run 16 stress 0.1258672 
## Run 17 stress 0.1358024 
## Run 18 stress 0.09036937 
## ... Procrustes: rmse 0.003376715  max resid 0.01503468 
## Run 19 stress 0.126985 
## Run 20 stress 0.1360016 
## *** Solution reached

plot_ordination(ps.inall.col, ord, type = "species", color = "colors") + 
  scale_color_manual(values =  c("salmon", "darkgreen", "cyan", "red", "blue", "plum")) +
  theme_bw() +
  theme(legend.position = "none")

0.6.2.3 visualisation

Далее идут одинаковые картинки по всем группам.

l_vst <- color_filt(ps.inall, submod_df)
l_vst

$blue \(blue\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 86 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 86 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 86 tips and 85 internal nodes ] refseq() DNAStringSet: [ 86 reference sequences ]

\(blue\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 86 89493 0 11387 1819 Avg#Reads 2556.94

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 86(100%) 86(100%) 86(100%) 85(98.84%) 79(91.86%) 50(58.14%) 7(8.14%)

Metadata variables: 4 SampleID, Day, Description, Group

\(blue\)heat \(blue\)heat_rel \(blue\)tree \(blue\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq314	Bacteria	Cyanobacteria	Vampirivibrionia	Obscuribacterales	Obscuribacteraceae	NA	NA
Seq23	Bacteria	Cyanobacteria	Vampirivibrionia	Obscuribacterales	Obscuribacteraceae	NA	NA
Seq65	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	NA
Seq188	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	soli
Seq334	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	NA
Seq75	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	NA
Seq167	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	massiliensis
Seq273	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Kaistiaceae	Kaistia	NA
Seq131	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Pseudolabrys	NA
Seq138	Bacteria	Proteobacteria	Alphaproteobacteria	Micropepsales	Micropepsaceae	NA	NA
Seq104	Bacteria	Proteobacteria	Alphaproteobacteria	Micropepsales	Micropepsaceae	NA	NA
Seq129	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Altererythrobacter	NA
Seq28	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Devosiaceae	Devosia	NA
Seq360	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Mesorhizobium	NA
Seq292	Bacteria	Proteobacteria	Alphaproteobacteria	Caulobacterales	Caulobacteraceae	NA	NA
Seq166	Bacteria	Proteobacteria	Alphaproteobacteria	Caulobacterales	Hyphomonadaceae	Hirschia	NA
Seq39	Bacteria	Gemmatimonadota	Gemmatimonadetes	Gemmatimonadales	Gemmatimonadaceae	NA	NA
Seq30	Bacteria	Actinobacteriota	Thermoleophilia	Solirubrobacterales	Solirubrobacteraceae	Conexibacter	NA
Seq151	Bacteria	Actinobacteriota	Thermoleophilia	Solirubrobacterales	67-14	NA	NA
Seq250	Bacteria	Actinobacteriota	Thermoleophilia	Solirubrobacterales	67-14	NA	NA
Seq135	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq46	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq35	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq106	Bacteria	Myxococcota	Polyangia	Polyangiales	Sandaracinaceae	NA	NA
Seq345	Bacteria	Bdellovibrionota	Oligoflexia	0319-6G20	NA	NA	NA
Seq198	Bacteria	Bdellovibrionota	Bdellovibrionia	Bdellovibrionales	Bdellovibrionaceae	Bdellovibrio	NA
Seq97	Bacteria	Bdellovibrionota	Bdellovibrionia	Bdellovibrionales	Bdellovibrionaceae	Bdellovibrio	NA
Seq183	Bacteria	Dependentiae	Babeliae	Babeliales	UBA12409	NA	NA
Seq424	Bacteria	Actinobacteriota	Actinobacteria	Propionibacteriales	Nocardioidaceae	Kribbella	NA
Seq136	Bacteria	Actinobacteriota	Acidimicrobiia	Microtrichales	Iamiaceae	Iamia	NA
Seq174	Bacteria	Actinobacteriota	Acidimicrobiia	Microtrichales	Iamiaceae	Iamia	NA
Seq43	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	Galbitalea	NA
Seq420	Bacteria	Actinobacteriota	Actinobacteria	Micromonosporales	Micromonosporaceae	Luedemannella	NA
Seq127	Bacteria	Actinobacteriota	Actinobacteria	Micromonosporales	Micromonosporaceae	Dactylosporangium	NA
Seq288	Bacteria	Actinobacteriota	Actinobacteria	Streptomycetales	Streptomycetaceae	NA	NA
Seq449	Bacteria	Actinobacteriota	Actinobacteria	Propionibacteriales	Propionibacteriaceae	Jiangella	NA
Seq91	Bacteria	Bacteroidota	Bacteroidia	Bacteroidales	NA	NA	NA
Seq149	Bacteria	Bacteroidota	Bacteroidia	Bacteroidetes VC2.1 Bac22	NA	NA	NA
Seq169	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	Sphingobacteriaceae	Mucilaginibacter	calamicampi
Seq81	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	NS11-12 marine group	NA	NA
Seq211	Bacteria	Bacteroidota	Bacteroidia	NA	NA	NA	NA
Seq119	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	env.OPS 17	NA	NA
Seq77	Bacteria	Spirochaetota	Spirochaetia	Spirochaetales	Spirochaetaceae	Salinispira	NA
Seq175	Bacteria	Spirochaetota	Spirochaetia	Spirochaetales	Spirochaetaceae	Spirochaeta 2	NA
Seq305	Bacteria	Chloroflexi	Anaerolineae	Anaerolineales	Anaerolineaceae	NA	NA
Seq379	Bacteria	Chloroflexi	Chloroflexia	Chloroflexales	Roseiflexaceae	NA	NA
Seq165	Bacteria	Chloroflexi	Chloroflexia	Chloroflexales	Roseiflexaceae	NA	NA
Seq327	Bacteria	Chloroflexi	Chloroflexia	Thermomicrobiales	JG30-KF-CM45	NA	NA
Seq252	Bacteria	Armatimonadota	Fimbriimonadia	Fimbriimonadales	Fimbriimonadaceae	NA	NA
Seq382	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Pedosphaerales	Pedosphaeraceae	NA	NA
Seq123	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Opitutales	Opitutaceae	Lacunisphaera	limnophila
Seq124	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Terrimicrobiaceae	Terrimicrobium	NA
Seq325	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Chthoniobacteraceae	LD29	NA
Seq272	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Verrucomicrobiales	Verrucomicrobiaceae	Roseimicrobium	gellanilyticum
Seq402	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Verrucomicrobiales	Rubritaleaceae	Luteolibacter	NA
Seq322	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	NA	NA
Seq356	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	NA	NA
Seq259	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	Pir4 lineage	NA
Seq279	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	Pir4 lineage	NA
Seq214	Bacteria	Planctomycetota	Planctomycetes	Planctomycetales	Rubinisphaeraceae	SH-PL14	NA
Seq209	Bacteria	Planctomycetota	Planctomycetes	Planctomycetales	Schlesneriaceae	Schlesneria	NA
Seq229	Bacteria	Acidobacteriota	Vicinamibacteria	Vicinamibacterales	Vicinamibacteraceae	NA	NA
Seq275	Bacteria	Acidobacteriota	Vicinamibacteria	Vicinamibacterales	Vicinamibacteraceae	NA	NA
Seq247	Bacteria	Acidobacteriota	Vicinamibacteria	Vicinamibacterales	NA	NA	NA
Seq316	Bacteria	Bacteroidota	Kapabacteria	Kapabacteriales	NA	NA	NA
Seq371	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq12	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq153	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq326	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq10	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	NA	NA
Seq82	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Terrimonas	NA
Seq462	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Pseudoflavitalea	NA
Seq150	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Flavitalea	NA
Seq290	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Edaphobaculum	NA
Seq339	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Edaphobaculum	NA
Seq120	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Taibaiella	NA
Seq233	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	211ds20	NA	NA
Seq133	Bacteria	Proteobacteria	Gammaproteobacteria	Steroidobacterales	Steroidobacteraceae	Steroidobacter	flavus
Seq349	Bacteria	Proteobacteria	Gammaproteobacteria	Steroidobacterales	Steroidobacteraceae	Steroidobacter	NA
Seq315	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq57	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq96	Bacteria	Proteobacteria	Gammaproteobacteria	R7C24	NA	NA	NA
Seq170	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq94	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Rhodanobacteraceae	Dokdonella	ginsengisoli
Seq114	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	NA	NA
Seq235	Bacteria	Proteobacteria	Gammaproteobacteria	Legionellales	Legionellaceae	Legionella	NA

$cyan \(cyan\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 30 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 30 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 30 tips and 29 internal nodes ] refseq() DNAStringSet: [ 30 reference sequences ]

\(cyan\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 30 176073 336 15108 4911 Avg#Reads 5030.66

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 30(100%) 30(100%) 30(100%) 30(100%) 30(100%) 29(96.67%) 2(6.67%)

Metadata variables: 4 SampleID, Day, Description, Group

\(cyan\)heat \(cyan\)heat_rel \(cyan\)tree \(cyan\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq98	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Burkholderiaceae	Burkholderia	telluris
Seq64	Bacteria	Proteobacteria	Alphaproteobacteria	Azospirillales	Inquilinaceae	Inquilinus	ginsengisoli
Seq4	Bacteria	Proteobacteria	Alphaproteobacteria	Azospirillales	Inquilinaceae	Inquilinus	NA
Seq244	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Pseudorhodoplanes	NA
Seq11	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Bradyrhizobium	NA
Seq74	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Bradyrhizobium	NA
Seq22	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Starkeya	NA
Seq45	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Allorhizobium	NA
Seq9	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Allorhizobium	NA
Seq29	Bacteria	Myxococcota	Polyangia	Nannocystales	Nannocystaceae	Nannocystis	NA
Seq239	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Labilithrix	NA
Seq50	Bacteria	Actinobacteriota	Actinobacteria	Corynebacteriales	Mycobacteriaceae	Mycobacterium	NA
Seq34	Bacteria	Actinobacteriota	Actinobacteria	Streptosporangiales	Streptosporangiaceae	Herbidospora	NA
Seq56	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq25	Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Terribacillus	NA
Seq3	Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	NA
Seq5	Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	NA
Seq13	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	NA	NA
Seq7	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Solibacillus	NA
Seq19	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Solibacillus	NA
Seq17	Bacteria	Planctomycetota	Planctomycetes	Isosphaerales	Isosphaeraceae	Singulisphaera	NA
Seq324	Bacteria	Acidobacteriota	Acidobacteriae	Acidobacteriales	Acidobacteriaceae (Subgroup 1)	Edaphobacter	NA
Seq16	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq6	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq1	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq109	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq49	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Taibaiella	NA
Seq73	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Spirosomaceae	Dyadobacter	NA
Seq15	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Rhodanobacteraceae	Luteibacter	NA
Seq26	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Luteimonas	NA

$darkgreen \(darkgreen\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 44 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 44 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 44 tips and 43 internal nodes ] refseq() DNAStringSet: [ 44 reference sequences ]

\(darkgreen\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 44 12360 0 2468 65 Avg#Reads 353.14

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 44(100%) 44(100%) 44(100%) 43(97.73%) 40(90.91%) 24(54.55%) 3(6.82%)

Metadata variables: 4 SampleID, Day, Description, Group

\(darkgreen\)heat \(darkgreen\)heat_rel \(darkgreen\)tree \(darkgreen\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq342	Archaea	Crenarchaeota	Nitrososphaeria	Nitrososphaerales	Nitrososphaeraceae	NA	NA
Seq196	Archaea	Crenarchaeota	Nitrososphaeria	Nitrososphaerales	Nitrososphaeraceae	Candidatus Nitrocosmicus	NA
Seq276	Archaea	Crenarchaeota	Nitrososphaeria	Nitrososphaerales	Nitrososphaeraceae	NA	NA
Seq419	Archaea	Crenarchaeota	Nitrososphaeria	Nitrososphaerales	Nitrososphaeraceae	NA	NA
Seq454	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	Aquicella	NA
Seq329	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	jaspsi
Seq332	Bacteria	Proteobacteria	Alphaproteobacteria	Rhodospirillales	Magnetospiraceae	NA	NA
Seq429	Bacteria	Proteobacteria	Alphaproteobacteria	Acetobacterales	Acetobacteraceae	NA	NA
Seq195	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Pseudorhodoplanes	NA
Seq144	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Pseudolabrys	NA
Seq369	Bacteria	Proteobacteria	Alphaproteobacteria	Micropepsales	Micropepsaceae	NA	NA
Seq362	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Altererythrobacter	NA
Seq428	Bacteria	Proteobacteria	Alphaproteobacteria	Caulobacterales	Caulobacteraceae	Phenylobacterium	NA
Seq347	Bacteria	Nitrospirota	Nitrospiria	Nitrospirales	Nitrospiraceae	Nitrospira	japonica
Seq328	Bacteria	Myxococcota	Polyangia	Haliangiales	Haliangiaceae	Haliangium	NA
Seq348	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq308	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq256	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Minicystis	NA
Seq302	Bacteria	Bdellovibrionota	Oligoflexia	0319-6G20	NA	NA	NA
Seq435	Bacteria	Actinobacteriota	Actinobacteria	Propionibacteriales	Propionibacteriaceae	Microlunatus	NA
Seq291	Bacteria	Actinobacteriota	Acidimicrobiia	IMCC26256	NA	NA	NA
Seq392	Bacteria	Actinobacteriota	Acidimicrobiia	Microtrichales	Ilumatobacteraceae	NA	NA
Seq321	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	NS11-12 marine group	NA	NA
Seq186	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq204	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Paenisporosarcina	NA
Seq146	Bacteria	Spirochaetota	Spirochaetia	Spirochaetales	Spirochaetaceae	Spirochaeta 2	NA
Seq359	Bacteria	Patescibacteria	Saccharimonadia	Saccharimonadales	LWQ8	NA	NA
Seq269	Bacteria	Chloroflexi	Chloroflexia	Chloroflexales	Roseiflexaceae	NA	NA
Seq267	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Opitutales	Opitutaceae	Lacunisphaera	NA
Seq320	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Opitutales	Opitutaceae	Opitutus	NA
Seq203	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Terrimicrobiaceae	Terrimicrobium	NA
Seq497	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	NA	NA
Seq384	Bacteria	Planctomycetota	Planctomycetes	Planctomycetales	NA	NA	NA
Seq155	Bacteria	Acidobacteriota	Blastocatellia	Blastocatellales	Blastocatellaceae	NA	NA
Seq350	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	env.OPS 17	NA	NA
Seq224	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Terrimonas	NA
Seq409	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Edaphobaculum	NA
Seq337	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Taibaiella	NA
Seq494	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Amoebophilaceae	Candidatus Amoebophilus	NA
Seq277	Bacteria	Proteobacteria	Gammaproteobacteria	NA	NA	NA	NA
Seq191	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq335	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Luteimonas	vadosa
Seq157	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Rhodanobacteraceae	Dokdonella	NA
Seq385	Bacteria	Proteobacteria	Gammaproteobacteria	Salinisphaerales	Solimonadaceae	NA	NA

$plum \(plum\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 30 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 30 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 30 tips and 29 internal nodes ] refseq() DNAStringSet: [ 30 reference sequences ]

\(plum\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 30 17172 0 3034 103 Avg#Reads 490.63

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 30(100%) 30(100%) 30(100%) 30(100%) 27(90%) 16(53.33%) 1(3.33%)

Metadata variables: 4 SampleID, Day, Description, Group

\(plum\)heat \(plum\)heat_rel \(plum\)tree \(plum\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq161	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	Aquicella	NA
Seq181	Bacteria	Proteobacteria	Gammaproteobacteria	CCD24	NA	NA	NA
Seq207	Bacteria	Cyanobacteria	Vampirivibrionia	Obscuribacterales	Obscuribacteraceae	NA	NA
Seq219	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	naasensis
Seq216	Bacteria	Proteobacteria	Alphaproteobacteria	Dongiales	Dongiaceae	Dongia	NA
Seq220	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	NA
Seq90	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Pseudolabrys	NA
Seq271	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	NA	NA
Seq111	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingobium	NA
Seq33	Bacteria	Proteobacteria	Alphaproteobacteria	Caulobacterales	Caulobacteraceae	Caulobacter	NA
Seq189	Bacteria	Actinobacteriota	Thermoleophilia	Solirubrobacterales	Solirubrobacteraceae	Solirubrobacter	NA
Seq85	Bacteria	Myxococcota	Polyangia	Polyangiales	BIrii41	NA	NA
Seq208	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	Galbitalea	NA
Seq218	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	Sphingobacteriaceae	Mucilaginibacter	NA
Seq215	Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	NA
Seq177	Bacteria	Fibrobacterota	Fibrobacteria	Fibrobacterales	Fibrobacteraceae	NA	NA
Seq375	Bacteria	Chloroflexi	Chloroflexia	Thermomicrobiales	JG30-KF-CM45	NA	NA
Seq430	Bacteria	Planctomycetota	Phycisphaerae	Phycisphaerales	Phycisphaeraceae	NA	NA
Seq368	Bacteria	Planctomycetota	Planctomycetes	Pirellulales	Pirellulaceae	NA	NA
Seq152	Bacteria	Planctomycetota	Planctomycetes	Gemmatales	Gemmataceae	Gemmata	NA
Seq304	Bacteria	Planctomycetota	Planctomycetes	Planctomycetales	NA	NA	NA
Seq141	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Terrimonas	NA
Seq343	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	NA	NA
Seq412	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Edaphobaculum	NA
Seq457	Bacteria	Proteobacteria	Gammaproteobacteria	Coxiellales	Coxiellaceae	Coxiella	NA
Seq130	Bacteria	Proteobacteria	Gammaproteobacteria	R7C24	NA	NA	NA
Seq346	Bacteria	Proteobacteria	Gammaproteobacteria	Salinisphaerales	Solimonadaceae	Alkanibacter	NA
Seq474	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	NA	NA
Seq472	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	NA	NA
Seq227	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	NA	NA

$red \(red\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 75 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 75 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 75 tips and 74 internal nodes ] refseq() DNAStringSet: [ 75 reference sequences ]

\(red\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 75 66650 241 5339 1528 Avg#Reads 1904.29

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 75(100%) 75(100%) 75(100%) 74(98.67%) 74(98.67%) 66(88%) 11(14.67%)

Metadata variables: 4 SampleID, Day, Description, Group

\(red\)heat \(red\)heat_rel \(red\)tree \(red\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq117	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Burkholderiaceae	Burkholderia	NA
Seq60	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Alcaligenaceae	NA	NA
Seq121	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Comamonadaceae	Variovorax	NA
Seq383	Bacteria	Proteobacteria	Alphaproteobacteria	Ferrovibrionales	Ferrovibrionaceae	Ferrovibrio	soli
Seq228	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	NA	NA
Seq178	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Beijerinckiaceae	Methylobacterium-Methylorubrum	NA
Seq171	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Beijerinckiaceae	Bosea	thiooxidans
Seq341	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Beijerinckiaceae	Bosea	NA
Seq89	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Tardiphaga	robiniae
Seq31	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Xanthobacteraceae	Starkeya	NA
Seq160	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Hyphomicrobiaceae	Hyphomicrobium	NA
Seq251	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingopyxis	NA
Seq373	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Altererythrobacter	NA
Seq108	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Mesorhizobium	NA
Seq71	Bacteria	Actinobacteriota	Thermoleophilia	Solirubrobacterales	Solirubrobacteraceae	Conexibacter	NA
Seq338	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Pajaroellobacter	NA
Seq230	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Pajaroellobacter	NA
Seq88	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Sorangium	NA
Seq303	Bacteria	Actinobacteriota	Acidimicrobiia	Microtrichales	Ilumatobacteraceae	NA	NA
Seq93	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Promicromonosporaceae	Promicromonospora	NA
Seq366	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	Leifsonia	NA
Seq134	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	NA	aoyamense
Seq411	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	Agromyces	NA
Seq115	Bacteria	Actinobacteriota	Actinobacteria	Corynebacteriales	Mycobacteriaceae	Mycobacterium	NA
Seq128	Bacteria	Actinobacteriota	Actinobacteria	Streptosporangiales	Streptosporangiaceae	Herbidospora	mongoliensis
Seq398	Bacteria	Actinobacteriota	Actinobacteria	Streptosporangiales	Streptosporangiaceae	Nonomuraea	NA
Seq78	Bacteria	Actinobacteriota	Actinobacteria	Streptosporangiales	Thermomonosporaceae	Actinocorallia	NA
Seq193	Bacteria	Actinobacteriota	Actinobacteria	Streptomycetales	Streptomycetaceae	Streptomyces	NA
Seq148	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	Sphingobacteriaceae	Pedobacter	NA
Seq307	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	NS11-12 marine group	NA	NA
Seq431	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	NS11-12 marine group	NA	NA
Seq205	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	lautus
Seq86	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq51	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq18	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq192	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq266	Bacteria	Firmicutes	Bacilli	Paenibacillales	Paenibacillaceae	Paenibacillus	NA
Seq278	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Domibacillus	NA
Seq99	Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	NA
Seq53	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Lysinibacillus	NA
Seq118	Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Lysinibacillus	NA
Seq76	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Terrimicrobiaceae	Terrimicrobium	NA
Seq232	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Terrimicrobiaceae	Terrimicrobium	NA
Seq168	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Terrimicrobiaceae	Terrimicrobium	NA
Seq387	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Chthoniobacteraceae	Chthoniobacter	NA
Seq285	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Chthoniobacterales	Chthoniobacteraceae	Chthoniobacter	NA
Seq217	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Verrucomicrobiales	Verrucomicrobiaceae	Verrucomicrobium	spinosum
Seq190	Bacteria	Planctomycetota	Planctomycetes	Gemmatales	Gemmataceae	Gemmata	NA
Seq249	Bacteria	Planctomycetota	Planctomycetes	Isosphaerales	Isosphaeraceae	Singulisphaera	NA
Seq254	Bacteria	Acidobacteriota	Acidobacteriae	Acidobacteriales	Acidobacteriaceae (Subgroup 1)	Edaphobacter	NA
Seq67	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq176	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq268	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Pseudoflavitalea	NA
Seq32	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Pseudoflavitalea	NA
Seq84	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Pseudoflavitalea	NA
Seq459	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	NA
Seq40	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	hibisci
Seq173	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	NA
Seq246	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	NA
Seq112	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Flavitalea	NA
Seq126	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	ginsengihumi
Seq102	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	arvensicola
Seq309	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	soli
Seq70	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq262	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq301	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Taibaiella	NA
Seq572	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	NA	NA
Seq436	Bacteria	Proteobacteria	Gammaproteobacteria	Steroidobacterales	Steroidobacteraceae	Steroidobacter	NA
Seq261	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq298	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq354	Bacteria	Proteobacteria	Gammaproteobacteria	Gammaproteobacteria Incertae Sedis	Unknown Family	Acidibacter	NA
Seq226	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Rhodanobacteraceae	Tahibacter	NA
Seq147	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Xanthomonas	NA
Seq370	Bacteria	Proteobacteria	Gammaproteobacteria	Diplorickettsiales	Diplorickettsiaceae	NA	NA
Seq486	Bacteria	Proteobacteria	Gammaproteobacteria	NA	NA	NA	NA

$salmon \(salmon\)ps phyloseq-class experiment-level object otu_table() OTU Table: [ 73 taxa and 35 samples ] sample_data() Sample Data: [ 35 samples by 3 sample variables ] tax_table() Taxonomy Table: [ 73 taxa by 7 taxonomic ranks ] phy_tree() Phylogenetic Tree: [ 73 tips and 72 internal nodes ] refseq() DNAStringSet: [ 73 reference sequences ]

\(salmon\)amp ampvis2 object with 5 elements. Summary of OTU table: Samples OTUs Total#Reads Min#Reads Max#Reads Median#Reads 35 73 115332 0 20534 779 Avg#Reads 3295.2

Assigned taxonomy: Kingdom Phylum Class Order Family Genus Species 73(100%) 73(100%) 73(100%) 72(98.63%) 71(97.26%) 66(90.41%) 12(16.44%)

Metadata variables: 4 SampleID, Day, Description, Group

\(salmon\)heat \(salmon\)heat_rel \(salmon\)tree \(salmon\)taxa

	Kingdom	Phylum	Class	Order	Family	Genus	Species
Seq79	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia	armeniaca
Seq122	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	NA	NA
Seq179	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	Pseudoduganella	NA
Seq145	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	Pseudoduganella	eburnea
Seq154	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia	NA
Seq222	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia	NA
Seq159	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Burkholderiaceae	Cupriavidus	NA
Seq8	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Burkholderiaceae	Cupriavidus	NA
Seq14	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Alcaligenaceae	Achromobacter	NA
Seq54	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Comamonadaceae	NA	paradoxus
Seq95	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Comamonadaceae	Xylophilus	NA
Seq340	Bacteria	Proteobacteria	Gammaproteobacteria	Burkholderiales	Comamonadaceae	Rhizobacter	NA
Seq264	Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	mucosissima
Seq333	Bacteria	Proteobacteria	Alphaproteobacteria	Reyranellales	Reyranellaceae	Reyranella	NA
Seq158	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Beijerinckiaceae	Microvirga	NA
Seq248	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Allorhizobium	NA
Seq245	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Devosiaceae	Devosia	neptuniae
Seq185	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Shinella	NA
Seq156	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	NA	NA
Seq42	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	NA	NA
Seq286	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Ensifer	NA
Seq21	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Allorhizobium	NA
Seq107	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Neorhizobium	NA
Seq231	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Allorhizobium	azooxidifex
Seq140	Bacteria	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	NA	NA
Seq378	Bacteria	Proteobacteria	Alphaproteobacteria	Caulobacterales	Caulobacteraceae	Phenylobacterium	mobile
Seq374	Bacteria	Myxococcota	Polyangia	Haliangiales	Haliangiaceae	Haliangium	NA
Seq257	Bacteria	Myxococcota	Polyangia	Polyangiales	Polyangiaceae	Pajaroellobacter	NA
Seq223	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Promicromonosporaceae	Cellulosimicrobium	NA
Seq83	Bacteria	Actinobacteriota	Actinobacteria	Micrococcales	Microbacteriaceae	Microbacterium	NA
Seq68	Bacteria	Actinobacteriota	Actinobacteria	Corynebacteriales	Mycobacteriaceae	Mycobacterium	NA
Seq293	Bacteria	Actinobacteriota	Actinobacteria	Glycomycetales	Glycomycetaceae	Glycomyces	NA
Seq72	Bacteria	Actinobacteriota	Actinobacteria	Streptomycetales	Streptomycetaceae	Streptomyces	NA
Seq101	Bacteria	Actinobacteriota	Actinobacteria	Streptomycetales	Streptomycetaceae	Streptomyces	NA
Seq265	Bacteria	Actinobacteriota	Actinobacteria	Streptomycetales	Streptomycetaceae	Streptomyces	NA
Seq260	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	NA	NA	NA
Seq180	Bacteria	Bacteroidota	Bacteroidia	Sphingobacteriales	Sphingobacteriaceae	Pedobacter	panaciterrae
Seq142	Bacteria	Verrucomicrobiota	Verrucomicrobiae	Verrucomicrobiales	Verrucomicrobiaceae	Roseimicrobium	NA
Seq163	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq253	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq116	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq184	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Microscillaceae	Ohtaekwangia	NA
Seq52	Bacteria	Bacteroidota	Bacteroidia	Flavobacteriales	Flavobacteriaceae	Flavobacterium	NA
Seq187	Bacteria	Bacteroidota	Bacteroidia	Flavobacteriales	Weeksellaceae	Chryseobacterium	ginsenosidimutans
Seq172	Bacteria	Bacteroidota	Bacteroidia	Flavobacteriales	Weeksellaceae	Chryseobacterium	NA
Seq283	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Flavitalea	NA
Seq113	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	NA
Seq110	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Niastella	NA
Seq132	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Pseudoflavitalea	NA
Seq37	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq162	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq182	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	humicola
Seq92	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq80	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	pinensis
Seq2	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq221	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq20	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq282	Bacteria	Bacteroidota	Bacteroidia	Chitinophagales	Chitinophagaceae	Chitinophaga	NA
Seq38	Bacteria	Bacteroidota	Bacteroidia	Cytophagales	Spirosomaceae	Dyadobacter	NA
Seq237	Bacteria	Proteobacteria	Gammaproteobacteria	Steroidobacterales	Steroidobacteraceae	Steroidobacter	agariperforans
Seq234	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Pseudoxanthomonas	NA
Seq59	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Stenotrophomonas	NA
Seq105	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Stenotrophomonas	NA
Seq344	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Lysobacter	NA
Seq41	Bacteria	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Lysobacter	NA
Seq243	Bacteria	Proteobacteria	Gammaproteobacteria	NA	NA	NA	NA
Seq406	Bacteria	Proteobacteria	Gammaproteobacteria	Legionellales	Legionellaceae	Legionella	NA
Seq139	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	NA
Seq24	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	NA
Seq27	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	NA
Seq87	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	NA
Seq55	Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	NA
Seq201	Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacterales	Enterobacteriaceae	Klebsiella	NA

mpd по стадиям

physeq_merged <- merge_samples(ps.f, "Group", fun=sum)

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

ps.f@sam_data

##                 Day Description  Group
## straw-16s-D01-1 D01       D01_1  early
## straw-16s-D01-2 D01       D01_2  early
## straw-16s-D01-3 D01       D01_3  early
## straw-16s-D03-1 D03       D03_1  early
## straw-16s-D03-2 D03       D03_2  early
## straw-16s-D03-3 D03       D03_3  early
## straw-16s-D05-1 D05       D05_1  early
## straw-16s-D05-2 D05       D05_2  early
## straw-16s-D05-3 D05       D05_3  early
## straw-16s-D05-4 D05       D05_4  early
## straw-16s-D05-5 D05       D05_5  early
## straw-16s-D07-2 D07       D07_2 middle
## straw-16s-D07-3 D07       D07_3 middle
## straw-16s-D08-1 D08       D08_1 middle
## straw-16s-D08-2 D08       D08_2 middle
## straw-16s-D08-3 D08       D08_3 middle
## straw-16s-D10-1 D10       D10_1 middle
## straw-16s-D10-2 D10       D10_2 middle
## straw-16s-D10-3 D10       D10_3 middle
## straw-16s-D10-4 D10       D10_4 middle
## straw-16s-D10-5 D10       D10_5 middle
## straw-16s-D12-1 D12       D12_1   late
## straw-16s-D12-2 D12       D12_2   late
## straw-16s-D12-3 D12       D12_3   late
## straw-16s-D13-1 D13       D13_1   late
## straw-16s-D13-2 D13       D13_2   late
## straw-16s-D13-3 D13       D13_3   late
## straw-16s-D14-1 D14       D14_1   late
## straw-16s-D14-2 D14       D14_2   late
## straw-16s-D14-3 D14       D14_3   late
## straw-16s-D14-4 D14       D14_4   late
## straw-16s-D14-5 D14       D14_5   late
## straw-16s-D15-1 D15       D15_1   late
## straw-16s-D15-2 D15       D15_2   late
## straw-16s-D15-3 D15       D15_3   late

# picante::mpd(l_vst$blue$ps@otu_table@.Data, cophenetic(l_vst$blue$ps@phy_tree)) %>% 
#   mean(na.rm = TRUE)
# 
# picante::mpd(l_vst$salmon$ps@otu_table@.Data, cophenetic(l_vst$salmon$ps@phy_tree)) %>% 
#   mean(na.rm = TRUE)

picante::ses.mpd(physeq_merged@otu_table@.Data, cophenetic(physeq_merged@phy_tree)) 

##        ntaxa  mpd.obs mpd.rand.mean mpd.rand.sd mpd.obs.rank  mpd.obs.z
## early    364 1.742096      1.862383  0.02930118            1 -4.1051776
## middle   490 1.839665      1.863938  0.02298162          147 -1.0561720
## late     822 1.877129      1.863599  0.01354403          842  0.9989395
##        mpd.obs.p runs
## early      0.001  999
## middle     0.147  999
## late       0.842  999

0.7 Add a metadata

list.files("meta/")

## [1] "cell_realtime_stat.xlsx" "cell_resp_ch_stat.xlsx" 
## [3] "period_legend.xlsx"

period_legend - соответствие номеров мешочков в 16S с днями, думаю лучше заменить везде обозначения 1, 3 и тд на дни cell_resp - данные по дыханию, по ним какую-нибудь простую статистику. Да, повторностей для эксперимента и контроля разное количество cell_realtime - это циклы выхода целлюлаз по реалтайму, я их нормировала по 16S, есть в этой же таблице. Что с ними делать особо не знаю, вроде вообще решили выкинуть

realtime.data <- readxl::read_excel("meta/cell_realtime_stat.xlsx")
period.data <- readxl::read_excel("meta/period_legend.xlsx")
resp.data <- readxl::read_excel("meta/cell_resp_ch_stat.xlsx")
realtime.data

## # A tibble: 36 × 29
##    id        day contr…¹ CELL_…² CELL_…³ CELL_…⁴ CELL_…⁵ CELL_…⁶ CELL_…⁷ CELL_…⁸
##    <chr>   <dbl>   <dbl>   <dbl>   <dbl>   <dbl>   <dbl>   <dbl>   <dbl>   <dbl>
##  1 G01-1-1     3    15.6    33.2    31.1    33.6    36.6    33.8    38.0    33.0
##  2 G01-1-2     3    15.5    33.3    31.3    32.9    36.2    33.8    41      32.7
##  3 G01-1-3     3    15.5    33.6    31.2    32.9    35.8    33.3    38.2    32.9
##  4 G01-2-1     3    17.1    36.0    31.1    33.1    36.9    36.4    38.8    33.8
##  5 G01-2-2     3    17.3    35.6    31.0    32.8    37.8    35.7    40.1    33.6
##  6 G01-2-3     3    17.1    36      31.1    32.8    38.1    35.2    37.6    33.6
##  7 G01-3-1     3    16.4    35.4    31.6    34.9    39.1    35.3    40.9    33.2
##  8 G01-3-2     3    16.5    35.6    31.3    34.4    37.9    34.6    38.9    33.1
##  9 G01-3-3     3    16.4    35.8    31.5    34.4    41.6    35.0    36.5    33.4
## 10 G05-1-1    28    18.2    26.3    29.1    29.4    28.2    28.4    37.4    34.5
## # … with 26 more rows, 19 more variables: CELL_193122 <dbl>, CELL_73229 <dbl>,
## #   CELL_47814 <dbl>, CELL_163125 <dbl>, CELL_73266 <dbl>, CELL_88582 <dbl>,
## #   CELL_63583 <dbl>, CELL_14199 <dbl>, CELL_95616 <dbl>, CELL_63504 <dbl>,
## #   CELL_08643 <chr>, CELL_199599 <dbl>, CELL_01426 <dbl>, CELL_71601 <dbl>,
## #   CELL_45099 <dbl>, CELL_191900 <dbl>, CELL_99463 <dbl>, CELL_74579 <dbl>,
## #   CELL_183403 <dbl>, and abbreviated variable names ¹​contr_16S, ²​CELL_172283,
## #   ³​CELL_203163, ⁴​CELL_83325, ⁵​CELL_188413, ⁶​CELL_109631, ⁷​CELL_188119, …

0.7.1 Realtime data

impute.mean <- function(x) replace(x, is.na(x), mean(x, na.rm = TRUE))

realtime_data <- realtime.data %>% 
  mutate(CELL_08643 = as.numeric(CELL_08643)) %>% 
  group_by(day) %>%
  mutate(nice_cell = impute.mean(CELL_08643)) %>% 
  mutate(day = as.factor(day))

## Warning in mask$eval_all_mutate(quo): NAs introduced by coercion

realtime_zjena <- readxl::read_excel("cellulases_gene_expression(1).xlsx")
geom_mean <-function(x){exp(mean(log(x)))}

realtime_zjena_geom <- realtime_zjena %>% 
  mutate(repeats = paste0(realtime_zjena$week, "-", rep(1:3, 3, each=3))) %>% 
  relocate(repeats, 1) %>% 
  group_by(repeats) %>% 
  summarise_if(is.numeric, geom_mean) %>% 
  mutate(week = as.factor(week)) %>% 
  arrange(week)

UPGMA целлюлаз(дистанция - брей).
Не очень понимаю, что это значит. В зависимости от дистанции получаются совсем разные кластеризации.

realtime_matrix <- realtime_zjena_geom %>% 
  column_to_rownames("repeats") %>% 
  select_if(is.numeric) %>% 
  as.matrix()

hcl <- hclust(vegan::vegdist(t(realtime_matrix), method="bray"), "average")
plot(hcl)

А вот - евклидова дистанция.
Ничего общего с предыдущей картинкой

hcl <- hclust(vegan::vegdist(t(realtime_matrix), method="euclidian"), "average")
plot(hcl)

Корплот целлюлаз.
Только корреляции

m = cor(realtime_matrix)
corrplot::corrplot(m)

Только достоверные корреляции
В общем - кластеры есть - но корреляцие недостоверные(за некоторыми исключениями)

cor_test_mat <- psych::corr.test(realtime_matrix)$p
corrplot::corrplot(m, p.mat = cor_test_mat, method = 'circle', type = 'lower', insig='blank',
         order = 'AOE', diag = FALSE)$corrPos -> p1

# text(p1$x, p1$y, round(p1$corr, 2))

То же, но матрица уже прологарифмирована.

cor_test_mat <- psych::corr.test(log(realtime_matrix))
corrplot::corrplot(cor_test_mat$r, p.mat = cor_test_mat$p, method = 'circle', type = 'lower', insig='blank',
         order = 'AOE', diag = FALSE)$corrPos -> p1

# text(p1$x, p1$y, round(p1$corr, 2))

add phylogenic tree (mafft - iqtree(ML))

realtime_tree <- ape::read.tree("al_chz.fasta.contree")
plot(realtime_tree)

library(tidyverse)

realtime_data %>% 
  select(c("day", "id", "contr_16S", "CELL_172283")) %>% 
  mutate(bio_repl = gsub("-[1-3]$", "", id)) %>% 
  group_by(bio_repl, day) %>% 
  summarise(contr_tmean = mean(contr_16S),
            data_tmean = mean(CELL_172283)) %>% 
  mutate(dCt = data_tmean - contr_tmean)

## `summarise()` has grouped output by 'bio_repl'. You can override using the
## `.groups` argument.

## # A tibble: 12 × 5
## # Groups:   bio_repl [12]
##    bio_repl day   contr_tmean data_tmean   dCt
##    <chr>    <fct>       <dbl>      <dbl> <dbl>
##  1 G01-1    3            15.5       33.4 17.8 
##  2 G01-2    3            17.2       35.9 18.7 
##  3 G01-3    3            16.4       35.6 19.2 
##  4 G05-1    28           18.4       26.2  7.81
##  5 G05-2    28           16.5       26.2  9.73
##  6 G05-3    28           18.5       32.0 13.6 
##  7 G10-1    91           17.4       26.2  8.72
##  8 G10-2    91           16.3       24.1  7.79
##  9 G10-3    91           17.5       26.3  8.81
## 10 G14-1    161          18.0       28.1 10.1 
## 11 G14-2    161          16.5       27.0 10.6 
## 12 G14-3    161          16.9       25.4  8.41

resp_data <- resp.data %>% 
  group_by(day) %>%
  mutate(control = impute.mean(control)) %>% 
  mutate(straw = impute.mean(straw)) 

resp_data

## # A tibble: 164 × 3
## # Groups:   day [27]
##      day control straw
##    <dbl>   <dbl> <dbl>
##  1     0    250   250 
##  2     3    285.  723.
##  3     3    263.  832.
##  4     3    131.  657.
##  5     3    307.  525.
##  6     3    241.  744.
##  7     3    245.  701.
##  8     3    245.  657.
##  9     7    274.  690.
## 10     7    296.  690.
## # … with 154 more rows

0.7.2 Knee plot

Дыхание - median(straw)/median(control)

Mожно использовать сторонний пакет(KneeArrower) что понять в каком месте knee_plot происходит перелом. Для элиминации повторностей возьмем медиану.   Я хз как этот пакет работает(ищет производную, но как сглаживает - хз, там матан), но он говорит что перелом происходит скорее на 60-80 днях.

(я исправил ошибки - стало ближе к твоим данным)

https://github.com/agentlans/KneeArrower - вот здесь можно почитать про матан

# resp_data %>% 
#   filter(!day == 0) %>% 
#   group_by(day) %>% 
#   summarise(median_control = median(control),
#             median_straw = median(straw)) %>% 
#   mutate(rel = median_straw/median_control) %>% 
#   ggplot() +
#   geom_point(aes(x = day, y = rel))

resp_median <- resp_data %>% 
  filter(!day == 0) %>% 
  group_by(day) %>% 
  summarise(median_control = median(control),
            median_straw = median(straw)) %>% 
  mutate(rel = median_straw/median_control)


thresholds <- c(0.25, 0.5, 0.75, 1)

# Find cutoff points at each threshold
cutoff.points <- lapply(thresholds, function(i) {
  findCutoff(resp_median$day, resp_median$rel, method="first", i)
})

x.coord <- sapply(cutoff.points, function(p) p$x)
y.coord <- sapply(cutoff.points, function(p) p$y)

# Plot the cutoff points on the scatterplot
plot(resp_median$day, resp_median$rel, pch=20, col="gray")
points(x.coord, y.coord, col="red", pch=20)
text(x.coord, y.coord, labels=thresholds, pos=4, col="red")

period_data <- period.data %>%
  mutate(bag_id = as.factor(bag_id))

period_data

## # A tibble: 15 × 2
##    bag_id   day
##    <fct>  <dbl>
##  1 1          3
##  2 2          7
##  3 3         14
##  4 4         21
##  5 5         28
##  6 6         35
##  7 7         49
##  8 8         63
##  9 9         77
## 10 10        91
## 11 11       105
## 12 12       119
## 13 13       140
## 14 14       161
## 15 15       182

0.7.3 альфа/дыхание

Ну я вот не очень понимаю что делать дальше - привязать кластеры к этой картинке?

resp_median_bags <- resp_median %>% 
  left_join(period.data, by="day") %>% 
  mutate(bag_id = as.factor(bag_id))

ps.f.r <- rarefy_even_depth(ps.f)

## You set `rngseed` to FALSE. Make sure you've set & recorded
##  the random seed of your session for reproducibility.
## See `?set.seed`

## ...

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

## 94OTUs were removed because they are no longer 
## present in any sample after random subsampling

## ...

## Found more than one class "phylo" in cache; using the first, from namespace 'phyloseq'

## Also defined by 'tidytree'

estimate_richness(ps.f.r) %>% 
  rownames_to_column("ID") %>% 
  mutate(bag_id = as.factor(
    as.numeric(
      gsub("\\..+","",
           gsub("straw\\.16s\\.D","", ID)
           )
      )
    )
    ) %>% 
  group_by(bag_id) %>% 
  summarise(Observed = mean(Observed),
            Shannon = mean(Shannon),
            InvSimpson = mean(InvSimpson)) %>% 
  left_join(period_data, by="bag_id") %>% 
  left_join(resp_median_bags, by="bag_id") %>% 
  mutate(
    Observed_scaled = scale(Observed),
    Shannon_scaled = scale(Shannon),
    InvSimpson_scaled = scale(InvSimpson),
    Respiration_scaled = scale(rel)
  ) %>% 
  select(c(bag_id, Observed_scaled, Shannon_scaled, InvSimpson_scaled, Respiration_scaled)) %>% 
  pivot_longer(c("Observed_scaled", "Shannon_scaled", "InvSimpson_scaled", "Respiration_scaled")) %>% 
  ggplot(aes(y = value, x = bag_id, group = name)) +
  geom_line(aes(color = name),
            alpha = 0.8) +
  theme_bw()

Корреляция -ртрицательная слабенькая, не особо достоверная и только Пирсона(которая работает для нормального распределения(у нас хроносерия, надо спирмана по хорошему))

alpha_resp <- phyloseq::estimate_richness(ps.f.r) %>% 
  rownames_to_column("ID") %>% 
  mutate(bag_id = as.factor(
    as.numeric(
      gsub("\\..+","",
           gsub("straw\\.16s\\.D","", ID)
           )
      )
    )
    ) %>% 
  group_by(bag_id) %>% 
  summarise(Observed = mean(Observed),
            Shannon = mean(Shannon),
            InvSimpson = mean(InvSimpson)) %>% 
  left_join(period_data, by="bag_id") %>% 
  left_join(resp_median_bags, by="bag_id") %>% 
  mutate(
    Observed_scaled = scale(Observed),
    Shannon_scaled = scale(Shannon),
    InvSimpson_scaled = scale(InvSimpson),
    Respiration_scaled = scale(rel)
  ) %>% 
  select(c(bag_id, Observed_scaled, Shannon_scaled, InvSimpson_scaled, Respiration_scaled))

cor.test(alpha_resp$Observed_scaled, alpha_resp$Respiration_scaled, method = "spearman")

## 
##  Spearman's rank correlation rho
## 
## data:  alpha_resp$Observed_scaled and alpha_resp$Respiration_scaled
## S = 230, p-value = 0.2629
## alternative hypothesis: true rho is not equal to 0
## sample estimates:
##        rho 
## -0.3939394

cor.test(alpha_resp$Shannon_scaled, alpha_resp$Respiration_scaled, method = "spearman")

## 
##  Spearman's rank correlation rho
## 
## data:  alpha_resp$Shannon_scaled and alpha_resp$Respiration_scaled
## S = 232, p-value = 0.2474
## alternative hypothesis: true rho is not equal to 0
## sample estimates:
##        rho 
## -0.4060606

cor.test(alpha_resp$InvSimpson_scaled, alpha_resp$Respiration_scaled, method = "spearman")

## 
##  Spearman's rank correlation rho
## 
## data:  alpha_resp$InvSimpson_scaled and alpha_resp$Respiration_scaled
## S = 226, p-value = 0.2956
## alternative hypothesis: true rho is not equal to 0
## sample estimates:
##       rho 
## -0.369697

cor.test(alpha_resp$Observed_scaled, alpha_resp$Respiration_scaled, method = "pearson")

## 
##  Pearson's product-moment correlation
## 
## data:  alpha_resp$Observed_scaled and alpha_resp$Respiration_scaled
## t = -2.7571, df = 8, p-value = 0.02479
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  -0.9223009 -0.1220128
## sample estimates:
##        cor 
## -0.6980158

cor.test(alpha_resp$Shannon_scaled, alpha_resp$Respiration_scaled, method = "pearson")

## 
##  Pearson's product-moment correlation
## 
## data:  alpha_resp$Shannon_scaled and alpha_resp$Respiration_scaled
## t = -2.8453, df = 8, p-value = 0.02164
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  -0.9255437 -0.1437787
## sample estimates:
##        cor 
## -0.7092031

cor.test(alpha_resp$InvSimpson_scaled, alpha_resp$Respiration_scaled, method = "pearson")

## 
##  Pearson's product-moment correlation
## 
## data:  alpha_resp$InvSimpson_scaled and alpha_resp$Respiration_scaled
## t = -2.2565, df = 8, p-value = 0.05401
## alternative hypothesis: true correlation is not equal to 0
## 95 percent confidence interval:
##  -0.899910986  0.009841998
## sample estimates:
##        cor 
## -0.6236489

0.8 Export all phyloseq object in one table

В табличке есть колонка cluster - “exl” в ней обозначает что это та часть датасета которая не пошла в WGCNA - мажоры в единичных семплах.

ps.m <- phyloseq::psmelt(ps.f)

ps.m <- ps.m %>% 
  mutate_if(is.character, as.factor) 

ps.data.out <- ps.m %>%
  select(-Group) %>% 
  pivot_wider(names_from = c(Day, Description, Sample), values_from = Abundance, values_fill = 0) 

#create empty dataframe with columnnames
external_empty_dataframe <- data.frame(OTU=factor(), cluster=factor(), stringsAsFactors = FALSE)

for (i in names(l_vst)) {
  a <- taxa_names(l_vst[[i]][["ps"]])
  b <- rep(i, length(a))
  d <- data.frame(OTU = as.factor(a),
                  cluster = as.factor(b))
  external_empty_dataframe <- rbind(external_empty_dataframe, d)
}

clusters.otu.df <- external_empty_dataframe
# add exl taxa -- taxa "exl"
d <- data.frame(OTU = as.factor(ps.exl.taxa),
                cluster = as.factor(rep("exl", length(ps.exl.taxa))))

clusters.otu.df <- rbind(clusters.otu.df, d)
ps.data.out.exl <- left_join(clusters.otu.df, ps.data.out, by="OTU")
# write.table(ps.data.out.exl, file = "ps.data.out.tsv", sep = "\t")
ps.data.out.exl

0.8.1 Несколько картинок по динамике предсталенности отдельных фил

Bdellovibrionota - хищники, индикатор развитого сообщества

ps.m %>% 
  filter(Phylum == "Bdellovibrionota") %>% 
  group_by(Description, Day) %>% 
  summarise(Bs = sum(Abundance)) %>% 
  ggplot() +
  geom_boxplot(aes(x = Day, y = Bs)) +
  theme_bw()

## `summarise()` has grouped output by 'Description'. You can override using the
## `.groups` argument.

Myxococcota - вроде бы тоже, но как нам известно могут быть целлулотитиками

ps.m %>% 
  filter(Phylum == "Myxococcota") %>% 
  group_by(Description, Day) %>% 
  summarise(Bs = sum(Abundance)) %>% 
  ggplot() +
  geom_boxplot(aes(x = Day, y = Bs)) +
  theme_bw()

## `summarise()` has grouped output by 'Description'. You can override using the
## `.groups` argument.

Археи появляются тоже на поздних стадиях - вообще я бы хотел бы опять развить тему важности азотного метаболизма на поздних стадиях разложения.
Оч хочется метагеном, но не этот.
Кроме того хочется отметить, что эти минорные группы возникают на D12

ps.m %>% 
  filter(Phylum == "Crenarchaeota") %>% 
  group_by(Description, Day) %>% 
  summarise(Bs = sum(Abundance)) %>% 
  ggplot() +
  geom_boxplot(aes(x = Day, y = Bs)) +
  theme_bw()

## `summarise()` has grouped output by 'Description'. You can override using the
## `.groups` argument.

Gammaproteobacteria - они треть кластера blue

ps.m %>% 
  filter(Class == "Gammaproteobacteria") %>% 
  group_by(Description, Day) %>% 
  summarise(Bs = sum(Abundance)) %>% 
  ggplot() +
  geom_boxplot(aes(x = Day, y = Bs)) +
  theme_bw()

## `summarise()` has grouped output by 'Description'. You can override using the
## `.groups` argument.

0.8.2 Боксплоты по филам

Все филы - представлененось логорифмирована по основанию 2
Отдельные точки - суммы абсолютных значений ридов по дням
Логорифмированы уже суммы, а не отдельные филотипы

#select only major phylums
top_phylum <- ps.m %>% 
  count(Phylum) %>% 
  arrange(desc(n)) %>% 
  top_n(10) %>% 
  pull(Phylum)

## Selecting by n

ps.m %>% 
  filter(Phylum %in% top_phylum) %>% 
  mutate(
    Phylum = as.character(Phylum),
    Class = as.character(Class),
    phylum = ifelse(Phylum == "Proteobacteria", Class, Phylum)
  ) %>% 
  group_by(Description, Day, phylum) %>% 
  filter(!is.na(phylum)) %>% 
  summarise(Bs = log2(sum(Abundance))) %>% 
  ggplot(aes(x = Day, y = Bs)) +
  geom_boxplot(fill="#4DBBD5B2", alpha=0.4) +
  theme_bw() +
  facet_wrap(~ phylum)

## `summarise()` has grouped output by 'Description', 'Day'. You can override
## using the `.groups` argument.

## Warning: Removed 46 rows containing non-finite values (stat_boxplot).